Project Summary It is increasingly evident that interspecies antagonism is intrinsic to life in the bacterial kingdom. The type VI secretion system (T6SS) is a complex intercellular protein delivery pathway. Though the system was initially thought to target host cells, during the prior award period our group discovered the primary function of the pathway is to deliver toxins in a cell contact-dependent manner between bacteria. Since this discovery, our group and others have learned that T6SS effectors are highly diverse. Superfamilies of effectors bearing little primary sequence identity have been described for toxins that target the peptide and carbohydrate components of the bacterial cell wall, membrane phospholipids, and DNA. Despite the potency of these toxins, many species encode multiple T6SS effectors, often with different mechanisms of action. The selective pressures driving the maintenance of diverse effector arsenals remain unknown. Additionally, while the primary function of the T6SS appears to be the delivery of toxins to competitor bacteria, several classes of effectors have been described that have targets conserved between bacteria and eukaryotes, such as phospholipids and nucleic acids. This raises the possibility that T6SS effectors could serve as a reservoir of biochemical functionality that contributes to the evolution of toxins employed by pathogens to target hosts. In this proposal, we examine both the functional consequences of effector diversity, and the connections between host and bacterial cell targeting toxins through the characterization of new effector mechanisms of action. In Aim 1, we propose a secretion-independent, novel quantitative mass spectrometry-based approach for defining new T6S effectors. Aim 2 focuses on the biochemical characterization of classes of effectors predicted by bioinformatics to act as ADP-ribosyltransferases, a mechanism common to many eukaryotic-targeting toxins. Finally, in Aim 3, we will employ a high throughput sequencing approach to test the hypothesis that a diverse effector repertoire promotes bacterial fitness in the face of varying environmental conditions that impact toxin efficacy. The studies outlined in this proposal stand to contribute significantly both to our basic understanding of the T6SS, and to the role that the pathway and its effectors play in bacterial pathogen fitness and evolution.